Spanish Human Proteome Project (sHPP).
Proyecto SRM CIMA.
María I. Mora, Fernando J. CorralesLaboratorio de Proteómica, CIMA, Universidad de Navarra
Proteína de interésSecuencia
Datos MS/MS
Crear Método MRM
Péptidos proteotípicos
Peptide verification
Crear Método MRM “optimizado”
Final Method Crear Método MRM “definitivo” (Scheduled)
Transiciones
Estrategia general
*Adquirir
*Evaluar
*Optimizar!!!
*Adquirir
*Evaluar
ensembl_gene_id external_gene_idACC Uniprot descriptionENSG00000167978 SRRM2 Q9UQ35 serine/arginine repetitive matrix 2 [Source:HGNC Symbol;Acc:16639]ENSG00000140750 ARHGAP17 Q68EM7 Rho GTPase activating protein 17 [Source:HGNC Symbol;Acc:18239]ENSG00000177238 TRIM72 Q6ZMU5 tripartite motif containing 72 [Source:HGNC Symbol;Acc:32671]ENSG00000090861 AARS P49588 alanyl-tRNA synthetase [Source:HGNC Symbol;Acc:20]ENSG00000135723 FHOD1 Q9Y613 formin homology 2 domain containing 1 [Source:HGNC Symbol;Acc:17905]ENSG00000166501 PRKCB P05771 protein kinase C, beta [Source:HGNC Symbol;Acc:9395]ENSG00000079616 KIF22 Q14807 kinesin family member 22 [Source:HGNC Symbol;Acc:6391]ENSG00000171490 RSL1D1 O76021 ribosomal L1 domain containing 1 [Source:HGNC Symbol;Acc:24534]ENSG00000177200 CHD9 Q3L8U1 chromodomain helicase DNA binding protein 9 [Source:HGNC Symbol;Acc:25701]ENSG00000166848 TERF2IP Q9NYB0 telomeric repeat binding factor 2, interacting protein [Source:HGNC Symbol;Acc:19246]ENSG00000070770 CSNK2A2 P19784 casein kinase 2, alpha prime polypeptide [Source:HGNC Symbol;Acc:2459]ENSG00000065457 ADAT1 Q9BUB4 adenosine deaminase, tRNA-specific 1 [Source:HGNC Symbol;Acc:228]ENSG00000015413 DPEP1 P16444 dipeptidase 1 (renal) [Source:HGNC Symbol;Acc:3002]ENSG00000166747 AP1G1 O43747 adaptor-related protein complex 1, gamma 1 subunit [Source:HGNC Symbol;Acc:555]ENSG00000141012 GALNS P34059 galactosamine (N-acetyl)-6-sulfate sulfatase [Source:HGNC Symbol;Acc:4122]ENSG00000100726 TELO2 Q9Y4R8 TEL2, telomere maintenance 2, homolog (S. cerevisiae) [Source:HGNC Symbol;Acc:29099]ENSG00000103381 CPPED1 Q9BRF8 calcineurin-like phosphoesterase domain containing 1 [Source:HGNC Symbol;Acc:25632]ENSG00000157106 SMG1 Q96Q15 smg-1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans) [Source:HGNC Symbol;Acc:30045]ENSG00000122299 ZC3H7A Q8IWR0 zinc finger CCCH-type containing 7A [Source:HGNC Symbol;Acc:30959]ENSG00000069345 DNAJA2 O60884 DnaJ (Hsp40) homolog, subfamily A, member 2 [Source:HGNC Symbol;Acc:14884]
20 proteínas asignadas a CIMA
Prueba preliminar en células Huh7
Preparación de la muestra
a) Lisis: en Urea 7M / Thiourea 2M / CHAPS 4% / DTT 40mM
b) Precipitación: cleanup kit (BioRad) Pellet resuspendido en Bicarbonato de Amonio 50mM
PROBLEMA: Resuspensión!!!!
c) Digestión: Reducción 10mM DTT, 56ºC, 30’
Alquilación 28mM IAA, RT, 20’
Digestión 1:50, 37ºC, o/n
d) A QTRAP 5500: 1 ug / run
Transiciones: Definidas por MRM pilot
3
2
4
Ignore if:
-KK/ KR/ RR/ RK (end)
-end of sequence
and
1
Métodos MRM individuales
Péptidos Transiciones pept - transic dwell time (msec) scan time (s)
A 20 - 101 15 2.01B 28 - 128 11 2.04C 23 -130 10 1.95
ARHGAP17 Q68EM7 10 55 1 A 10 - 55 30 1.92TRIM72 Q6ZMU5 11 61 1 A 11 - 61 28 2.01AARS P49588 20 109 1 A 20 - 109 13 1.96
A 13 -71 23 1.98B 17 - 90 18 2.06
PRKCB P05771 13 73 1 A 13 - 73 20 1.82KIF22 Q14807 18 98 1 A 18 - 98 15 1.96
RSL1D1 O76021 10 52 1 A 10 -52 32 1.91A 15 -80 20 1.99B 21 -112 13 2.01C 19 -98 15 1.96
TERF2IP Q9NYB0 12 68 1 A 12 -68 25 1.97CSNK2A2 P19784 9 48 1 A 9 - 48 35 1.91
ADAT1 Q9BUB4 13 71 1 A 13 - 71 23 1.98DPEP1 P16444 10 59 1 A 10 - 59 29 2.01AP1G1 O43747 10 55 1 A 10 - 55 30 1.92GALNS P34059 11 60 1 A 11 - 60 28 1.97
A 14 - 75 20 1.87B 12 -65 25 1.94
CPPED1 Q9BRF8 8 49 1 A 8 - 49 35 1.95A 20 - 105 14 1.99B 23 - 116 13 2.08C 21 - 110 14 2.08D 19 - 97 16 2.03
ZC3H7A Q8IWR0 20 110 1 A 20 - 110 13 1.97DNAJA2 O60884 7 39 1 A 7 -39 45 1.94
PROTEINA Nº métodos
SRRM2 Q9UQ35 71 359 3
SMG1 Q96Q15
FHOD1 Q9Y613
CHD9 Q3L8U1
TELO2 Q9Y4R8
30 97
55 290
26 140
83 428
3
2
2
4
Condiciones LC
Pickup: 2ul
Loading: 8min (3ul/min)
Gradiente: 60 min (300nl/min)
Ejemplos
MRMpilotAnalyst
Proteina:
AARS
Peptido:
ALNEALK / 379.72
TR:
22.4’
+MRM (109 pairs): Exp 1, 22.400 min from Sample 1 (PepVerif_AARS_A_60min_1ul_2012 15 06) of PepVerif_AARS_A_60min_1ul_20... Max. 5.0e4 cps.
685.040/1070.600685.040/1141.640
514.030/686.360379.720/574.320
379.720/687.400379.720/612.340
379.720/499.250379.720/460.280
379.720/428.210438.720/761.420
438.720/575.310438.720/662.350
438.720/730.340
Q1/Q3 Masses, Da
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
4.6e4
4.8e4
5.0e4
Inte
ns
ity, c
ps
y5
y6 b6
b5
y4
b4
Programadas: 6
Observadas: 6
Programadas: 6
Observadas: 5
AnalystMRMpilot
Proteina:
TRIM72
Peptido:
ILEAHVEAK / 505.29
TR:
20.6’
+MRM (61 pairs): Exp 1, 20.605 min from Sample 1 (PepVerif_TRIM72_A_60min_1ul_2012 15 06) of PepVerif_TRIM72_A_60min_1ul... Max. 8036.0 cps.
553.990/927.580 505.290/654.360 505.290/783.400 337.190/654.360 505.290/896.480 505.290/863.460 505.290/583.320Q1/Q3 Masses, Da
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
Inte
ns
ity, c
ps
*
y6
y7
y8
b8
y5
Analyst
MRMpilot
Proteina:
TELO2
Peptido:
FDRPLVTFDLLGEDQLVLGR / 768.42
TR:
40.34’
Programadas: 5
Observadas: 5
+MRM (65 pairs): Exp 1, 40.311 min from Sample 1 (PepVerif_TELO2_B_60min_1ul_2012 15 06) of PepVerif_TELO2_B_60min_1ul_2... Max. 2800.0 cps.
593.980/1067.600890.470/1154.540
768.420/1212.690768.420/800.460
768.420/1099.610768.420/929.510
768.420/986.530609.360/791.460
609.360/920.500406.570/791.460
609.360/1033.580609.360/1146.670
Q1/Q3 Masses, Da
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
Inte
ns
ity, c
ps
y10
y7
y9
y8
y11
Proteina:
SRRM2
Peptido:
STTPAPK / 351.19
TR:
16.24’
Programadas: 5
Observadas: 5
Analyst
+MRM (101 pairs): Exp 1, 16.226 min from Sample 1 (PepVerif_SRRM2_A_60min_1ul_2012 15 06) of PepVerif_SRRM2_A_60min_1ul_... Max. 9.5e4 cps.
343.680/426.200789.940/959.570
789.940/1056.620526.960/959.570
526.960/734.420789.940/1185.660
351.190/412.260351.190/513.300
351.190/614.350351.190/555.280
351.190/458.220679.790/897.390
679.790/810.360679.790/1012.420
679.790/1083.450679.790/1170.490
Q1/Q3 Masses, Da
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e4
Inte
ns
ity, c
ps
y4
y5
y6
b6
b5
AnalystMRMpilot
Proteina:
CHD9
Peptido:
TEEQVQK / 431.22
TR:
21.65’
Programadas: 5
Observadas: 4
+MRM (80 pairs): Exp 1, 21.650 min from Sample 1 (PepVerif_CHD9_A_60min_1ul_2012 15 06) of PepVerif_CHD9_A_60min_1ul_201... Max. 5.8e4 cps.
980.520/1229.700654.010/1130.630
654.010/1229.700980.520/1191.600
431.220/631.340431.220/502.300
431.220/760.380431.220/715.330
431.220/587.270421.720/581.330
421.720/728.400421.720/696.340
421.720/609.300421.720/496.220
476.230/589.260317.820/589.260
476.230/717.350
Q1/Q3 Masses, Da
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
5.8e4
Inte
ns
ity, c
ps
b6
b5
y4y5
! !
Proteina:
ARGHAP17
Peptido:
DPVSAAVPAPGR / 568.81
TR:
19.85’ – 21.41’
MRMpilotAnalyst
+MRM (55 pairs): Exp 1, 19.849 min from Sample 1 (PepVerif_ARHGAP17_A_60min_1ul_2012 15 06) of PepVerif_ARHGAP17_A_60... Max. 8367.0 cps.
488.260/616.290568.810/1021.580
568.810/667.390568.810/738.430
379.540/497.280568.810/825.460
568.810/924.530446.230/463.230
446.230/578.250446.230/679.300
446.230/778.370
Q1/Q3 Masses, Da
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8367
Inte
ns
ity, c
ps
y11
y7
y8
y5
y9
y10
+MRM (55 pairs): Exp 1, 21.413 min from Sample 1 (PepVerif_ARHGAP17_A_60min_1ul_2012 15 06) of PepVerif_ARHGAP17_A_60m... Max. 2.3e4 cps.
488.260/663.330488.260/791.390
488.260/862.430488.260/829.410
488.260/715.360488.260/616.290
568.810/1021.580568.810/667.390
568.810/738.430379.540/497.280
568.810/825.460568.810/924.530
446.230/463.230446.230/578.250
Q1/Q3 Masses, Da
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
2.3e4
Inte
ns
ity, c
ps
y11
y7
y8
y5y9
y10Programadas: 6
Observadas: 6
Analyst
Proteina:
DNAJA2
Peptido:
ENEVFQR /
472.73
TR:
16.3’ – 25.5’
+MRM (39 pairs): Exp 1, 16.312 min from Sample 1 (PepVerif_DNAJA2_A_60min_1ul_2012 15 06) of PepVerif_DNAJA2_A_60min_1ul... Max. 2.2e4 cps.
880.800/1127.670660.850/1224.720 472.730/815.420 472.730/549.310 472.730/678.360 315.490/549.310 472.730/770.350 472.730/642.290 931.940/1226.580931.940/1000.480Q1/Q3 Masses, Da
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
Inte
ns
ity, c
ps
y6
y5
y4
b6y4*
b5
+MRM (39 pairs): Exp 1, 25.533 min from Sample 1 (PepVerif_DNAJA2_A_60min_1ul_2012 15 06) of PepVerif_DNAJA2_A_60min_1u... Max. 3756.0 cps.
880.800/1070.650880.800/1127.670
660.850/1224.720472.730/815.420
472.730/549.310472.730/678.360
315.490/549.310472.730/770.350
472.730/642.290931.940/1226.580
931.940/1000.480931.940/1129.530
Q1/Q3 Masses, Da
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3756
Inte
ns
ity, c
ps
y6
y4
y5
b6
b5
*
Programadas: 6
Observadas: 6 / 5
E en N-terminal
Resultados MRM (peptide verification- I)
Péptidos Transiciones Posibles péptidos num. transiciones num. transiciones programados programadas a optimizar (Huh7) observadas programadas
379.72 22.4 6 6365.2 20.7 / 22.69 2 / 5 5
738.88 37.35 / 36.18 4 / 6 7530.76 / 354.18 32.91 4 6
450.78 21.02 5 5439.24 27.15 5 6530.25 19.4 5 6407.72 20.13 / 21.2 5 / 5 5423.25 22.7 6 6430.72 27.5 5 6432.23 19.95 5 5471.24 28.24 5 5
568.81 / 379.54 19.85 / 21.41 6 / 6 6616.33 / 411.22 20.99 4 5
551.8 / 368.2 28.6 5 5493.77 20.46 / 20.77 6 / 6 6422.76 17.77 6 6429.74 18.1 5 5424.7 23.08 / 26.73 3 / 3 5
431.22 21.65 5 5462.72 / 308.82 16.7 6 7
372.7 16.94 / 18.86 / 20.37 5 / 2 / 4 5539.3 24.4 4 5
503.26 / 335.85 27.75 5 6386.71 19.47 5 5722.77 28.39 4 5
478.78 / 319.52 21.4 / 26.8 / 27.3 5 / 4 / 2 6
Q1 (z2 / z3) TR observadoProteína
55 290 11
10 55 3
10 55 4
20 109 5
13 71 4
CHD9
AARS
ADAT1
AP1G1
ARHGAP17
Resultados MRM (peptide verification- II)
Péptidos Transiciones Posibles péptidos num. transiciones num. transiciones programados programadas a optimizar (Huh7) observadas programadas
CPPED1 8 49 1 551.28 / 367.86 28.05 7 7932.93 / 622.29 32.25 3 5
351.71 16.28 5 5725.4 / 483.94 19.86 5 6
762.91 / 508.94 35.3 5 6886.45 / 591.3 29.38 4 5
472.73 / 315.49 16.3 / 25.5 6 / 5 6569.29 / 379.86 19.1 7 7
411.71 22.9 5 5464.25 / 309.83 17.3 6 6488.74 / 326.16 16.47 6 6536.78 / 358.19 25.51 5 6661.34 / 441.23 26.02 5 5
454.23 15.8 5 5453.76 / 302.84 23.15 5 6506.27 / 337.85 16.5 6 6
436.25 21.45 5 5351.22 19.5 4 5438.72 15.4 / 16.86 6 / 6 6422.71 18.16 / 20.68 6 / 6 6
782.76 / 587.32 32.16 4 5435.23 20.2 5 6
798.45 / 532.64 28.62 4 5414.22 22.1 4 6434.78 16.85 5 5
PRKCB 13 73 0
Proteína Q1 (z2 / z3) TR observado
10 52 2
11 60 3
18 98 3
10 59 3
30 97 5
9 48 5
7 39 3
FHOD1
GALNS
KIF22
RSL1D1
CSNK2A2
DNAJA2
DPEP1
Resultados MRM (peptide verification- III)
Péptidos Transiciones Posibles péptidos num. transiciones num. transiciones programados programadas a optimizar (Huh7) observadas programadas
396.24 20.18 / 22.14 / 22.18 3 / 3 / 3 5453.75 24.97 5 5579.29 19.05 5 5404.24 19.26 5 5376.2 18.95 5 5
370.24 16.55 5 5552.78 / 368.86 25.48 2 5
429.74 20.66 6 6598.8 17.22 5 5
615.81 / 410.87 27.41 5 6493.29 / 329.19 27.11 5 5
449.25 29.05 4 5920.95 / 614.3 34.1 5 5
393.7 15.81 4 4351.19 16.24 5 5
464.25 / 309.84 21.54 2 5759.02 / 569.52 35.18 2 4
389.2 17.15 2 4460.72 / 307.48 16.26 5 6
351.18 15.66 / 19.26 5 / 4 5414.75 17.52 5 5
650.86 / 434.25 28.44 4 6486.27 16.3 4 6467.75 19.8 5 5
729.4 / 486.6 24.2 4 5438.77 25.5 3 5540.37 27.52 4 6768.42 40.34 5 5510.77 27.5 3 5
TERF2IP 12 68 0TRIM72 11 61 1 505.29 / 337.19 20.59 5 6
369.19 15.4 / 18.4 5 / 6 6433.71 15.6 5 5
594.81 / 396.88 28.05 5 6417.76 17.98 5 5
465.77 / 310.85 23.17 6 6670.82 / 447.55 16.14 4 6
Q1 (z2 / z3) TR observado
20 110 6
Proteína
71 359 12
26 140 5
83 428 12SMG1
SRRM2
TELO2
ZC3H7A
Proteína de interésSecuencia
Datos MS/MS
Crear Método MRM
Péptidos proteotípicos
Peptide verification
Crear Método MRM “optimizado”
Final Method Crear Método MRM “definitivo” (Scheduled)
Transiciones
Estrategia general
*Adquirir
*Evaluar
*Optimizar!!!
*Adquirir
*Evaluar
Condiciones QTRAP 5500
MRM- Peptide Verification
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